Contributors: Guilia Weissenberger, Bas Lemmens, and Cliff Mathisen
How does the VitroJet deal with the air-water interface?
What is the air-water interface and why is it important?
“Interfacial water is not thin ‘water.’ In the steep water density gradient present at the air-water interface, extreme anisotropy coexists with a hydrogen-bonding network constrained by the lack of inversion symmetry. These features give rise to unprecedented, unanticipated and often unimagined phenomena from what we know about bulk water. The composition of interfacial layers can also be very different from that of the bulk solutions beneath.” – Dr. Agusitin J Collusi (CalTech)
The air-water interface is essentially the chaotic boundary where a solution meets the air. In CryoEM this is heavily speculated upon as the surface to volume ratio of a sample is extremely high following its deposition on a grid. According to a number of papers[2], macromolecules can behave differently at the AWI as hydrophobic groups can stick and cause the macromolecule to fall apart. In grid preparation, the sample is spread over a 3mm grid with a thickness of less than 100nm. That leaves a large part of the sample vulnerable to the interface before being vitrified. It presents one of the most difficult problems in CryoEM sample prep where numerous solutions are explored to keep your macromolecule “happy” on the grid. Detergents, custom-made grids as well as speed have been tried and tested to keep molecules in an intact and randomly distributed state.
Can speed mitigate the effects of the air-water interface?
Can the effects of the air-water interface (AWI) be outrun? Unless you are shielding your liquid from the surrounding air altogether, it’s unlikely. Molecules will always have some interactions as long as they are exposed. But how much exposure is needed to outrun this “deadly touch”? Let’s look at the timescales in cellular biology. If we consider free movement across a cell, a protein needs about 10 ms to travel the distance within a 1 µm E.coli bacterial cell.
In SPA CryoEM, 50 nm thin layers (20x smaller than an E.coli) consisting of buffer (water, diffusion coefficient approximately 10x higher) rather than complex intracellular environment are prepared. Looking at the calculation, a 10-20nm protein will likely migrate from one side to the other side of our thin film in less than 50 microseconds. It has also been calculated that a 100kDa molecule will have 1000 interactions with the interface of 100nm in a second [4]. That gives a particle plenty of time to fall apart at the interface. With these timescales in mind, it is difficult to imagine mechanics that can outrun particles’ Brownian motion in the microsecond time scale. In the mechanical setup of a sample preparation device there generally needs to be space between the deposition area and the dry, cold, cryogenic area. This sets a high bar for engineers in the field looking to outrun particles’ motion to the AWI. Furthermore, would all samples benefit from speed? That opens a larger discussion altogether as solutions have been tried exploring different aspects for particle stability. Certainly, some samples will benefit from speed but others will need some incubation time on the grid.
This simple illustration shows what may happen to particles immediately after deposition (top) and then perhaps a few milliseconds later (bottom). These images look similar but notice the higher concentration of particles on the AWI (light blue) on the bottom image compared to the top. Since this reaction occurs so quickly, it’s unlikely that freezing the sample after deposition will ever be fast enough to “outrun” this effect.What else can we do to reduce detrimental interface effects?
In the entire purification to structure determination workflow, grid preparation is only a small step, and getting good grids highly depends on the quality of the solution you deposit. Optimization is key in structural biology [1]. In many cases, the problems we image in our microscopes today, can be solved by further optimizing our biochemistry. After all, when isolating macromolecules, we do change the environmental conditions for the molecule significantly. Purification methods may prove to be less gentle than we anticipate. In current grid preparation technologies, it is difficult to see the effect of the changes made when optimizing sample conditions. The irreproducibility and variability encountered with the devices makes the process tedious and, often, frustrating. Furthermore, blotting is a crude technique when working to create layers at the nanometer scale. Sometimes, good grids can come out of sheer luck with the preparation device. Of course, this is not always the case, especially with highly stable and known complexes good results can be obtained. Although, overall, the success rate of grid preparation is considered low, with an average of 10 – 50% grids considered data collection worthy after having optimized sample conditions. This reinforces our idea that control needs to be given back to the scientists. When the variability of grid preparation is removed, the changes implemented can be seen quickly and efficiently.
How to obtain control in CryoEM sample preparation
Automation will play a key function. When reproducibility is achieved grid after grid and day after day, results will be easier to get as well as to interpret. This is exactly what the VitroJet will offer: control over each parameter to obtain consistency. This includes the unique deposition technique (coming up in a next post!) that does not require any additional wicking. A highly automated system handling pre-clipped autogrids without manual intervention from the user lets scientists focus primarily on the sample conditions. This will be imperative as they achieve the conditions to prevent the particle from getting stuck or falling apart at the air-water interface.
Do you think the air-water interface influences your research? Email the author to discuss this topic further.
References
- CARRAGHER, B., CHENG, Y., FROST, A., GLAESER, R. M., LANDER, G. C., NOGALES, E., & WANG, H. ‐W. (2019). Current outcomes when optimizing ‘standard’ sample preparation for single‐particle cryo‐EM. Journal of Microscopy. https://doi.org/10.1111/jmi.12834
- Chen, J., Noble, A. J., Kang, J. Y., & Darst, S. A. (2019). Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO. Journal of Structural Biology: X, 1(February), 100005. https://doi.org/10.1016/j.yjsbx.2019.100005
- D’Imprima, E., Floris, D., Joppe, M., Sánchez, R., Grininger, M., & Kühlbrandt, W. (2019). Protein denaturation at the air-water interface and how to prevent it. ELife. https://doi.org/10.7554/elife.42747
- Naydenova, K., & Russo, C. J. (2017). Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy. Nature Communications. https://doi.org/10.1038/s41467-017-00782-3
- Noble, A. J., Wei, H., Dandey, V. P., Zhang, Z., Tan, Y. Z., Potter, C. S., & Carragher, B. (2018). Reducing effects of particle adsorption to the air–water interface in cryo-EM. Nature Methods. https://doi.org/10.1038/s41592-018-0139-3
- Taylor, K. A., & Glaeser, R. M. (2008). Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. Journal of Structural Biology. https://doi.org/10.1016/j.jsb.2008.06.004